Molecular cytogenetics investigations utilizing fluorescence in situ hybridization (FISH) and digital imaging analysis has resulted in significant progress in cancer molecular cytogenetics, mapping of viral sequences and single-copy cancer-related genes. Kaposi's sarcoma (KS) is a neoplasia of rising incidence due to it's association with infection by human immunodeficiency virus and AIDS. Until recently, the only cultured KS cells available were hyperplastic normal diploid cells. Two tumorigenic KS cell lines, one established from a patient with AIDS and another derived from a patient with renal transplant-associated KS who also had an acute infection with cytomegalovirus, have been isolated and were examined cytogenetically. Deletion and translocation of the short arm of chromosome 3 at region 3p14 was identified in both cell lines. Nonrandom alterations in these lines may be a key contributing lesion to the process of neoplastic development, and thus provide a cytogenetic marker leading to the isolation of specific molecular probes for detecting tumor cells in KS. In gene therapy, the delivery system is critical for a successful outcome. The ability of wild type adeno- associated virus (AAV) to integrate site-specifically into chromosome 19 and to infect a variety of cell types, including nondividing cells, makes this agent unique for gene therapy. To examine if AAV recombinants retained the site-specific integration associated with the wild type virus, the integration sites were determined by FISH. FISH analysis of HeLa clones transfected with recombinant AAV demonstrated that in contrast to the wild type virus, the recombinant virus did not specifically integrate in chromosome 19, but integrated nonrandomly to regions of proto-oncogenes. These results are important in evaluating a delivery system for gene therapy. Localization of genes in the human genome has been essential for advances in the molecular characterization of chromosome alterations and understanding of their role in cancer development. The direct visualization of the fluorescent signal on banded metaphase chromosomes is critical to the resolution of mapping. A procedure was developed in our laboratory based on contrast enhanced digital images of DAPI banded chromosomes. With this procedure immune gamma interferon, HER4/erbB-4, Syrian hamster Tp53 and human macrophage metalloelastase gene have been mapped. The localization of these genes will facilitate linkage analysis, identification of gene interactions as well as the analysis of specific cancer cell chromosomal rearrangements involving their loci.